There is a growing need for analytical methods to identify and quantify the interaction of low molecular weight drug compounds with biological membranes. The use of lipid bilayer arrays has the potential to provide a cheap and efficient high- throughput analytical method capable of addressing these issues. There are several key issues which need to be resolved if lipid microarrays are to achieve the level of success obtained with DNA microarrays assays. Chief among these is the need for a noninvasive method to detect drug association to the lipid microarray surface and the subsequent perturbation of the lipid matrix due to drug interaction. The nonlinear techniques of ultraviolet-visible sum-frequency generation (UV-Vis SFG) and infrared- visible sum-frequency generation (IR-Vis SFG) imaging may hold the answer to this problem. Several attributes of nonlinear imaging make it a promising method for detecting drug-membrane interaction on microarrays, including the ability to quantify the detected signal, the high optical resolution and the inherent surface specificity. The crucial first steps to implementing UV-Vis SFG and IR-Vis SFG imaging for the investigation of drug association on lipid microarrays have already been achieved. Specific Aim #1 will focus on the development of UV-Vis SFG for the detection of low molecular weight molecules in planar supported lipid bilayers (PSLBs). The use of UV- Vis SFG for measuring protein adsorption to membranes, which is an extension of the goals of the previous grant period, will also be pursued. The nonlinear spectral response from several model protein and drug targets will be investigated in an attempt to more fully understand the factors controlling their detection by UV-Vis SFG in Aim #1. The application of UV-Vis SFG for the screening of potential ion channel inhibitors is explored in Aim #2. Examination of the influence of drugs on the thermotropic phase transition and phase behavior of lipid membranes by IR-Vis SFG spectroscopy and IR- Vis SFG high-throughput imaging is explored in Aim #3. These studies are designed to demonstrate the utility of nonlinear imaging methods in conjunction with micropatterned fluid lipid bilayer arrays for high-throughput drug screening applications. PUBLIC HEALTH RELEVANCE: Membrane proteins, either associated or integral, account for up to two thirds of known drug targets. The partitioning of drugs into the hydrophobic environment of the lipid membrane controls the bioavailability of these materials. The goals of the proposed studies are to develop analytical tools for the noninvasive, specific spectroscopic detection of proteins and small molecule (drug) interactions with biological membranes. The practical implementation of these technologies will have a significant impact on the pharmacological screening of drug candidates, and the investigation of drug-membrane interactions.